Invertase inhibitor

ABSTRACT

This invention relates to a nucleic acid that contains at least one nucleic acid sequence coding for a polypeptide, polypeptide being capable of reducing the enzymatic activity of an invertase; the polypeptide itself; and transgenic plants that contain this nucleic acid sequence. The invention further relates to methods of preparing such transgenic plants having reduced storage sucrose loss.

This invention relates to a nucleic acid that contains at least onenucleic acid sequence coding for a polypeptide, said polypeptide beingcapable of reducing the enzymatic activity of an invertase, thepolypeptide itself, as well as transgenic plants that contain thisnucleic acid sequence. The invention further relates to methods ofpreparing such transgenic plants with reduced storage sucrose loss.

BACKGROUND OF THE INVENTION

During the storage of sugar beets (Beta vulgaris), in the period betweenharvest and processing, respiration or sucrose metabolism leads to asucrose loss of roughly 0.02% per day. This loss is further accompaniedby a significant diminution of quality as a consequence of the increaseof reducing sugars, in particular fructose and glucose (Burba, M.(1976), “Respiration and Sucrose Metabolism of Sugar Beets in Storage,”Zeitschrift für die Zuckerindustrie 26:647-658). The first metabolicstep in the breakdown of sucrose during the storage of beets isenzymatic hydrolysis by a vacuolar invertase. This enzyme is synthesizedde novo in the beet tissue after injury (Milling, R. J., Leigh, R. A.,and Hall, J. L. (1993), “Synthesis of a Vacuolar Acid Invertase inWashed Discs of Storage Root Tissue of Red Beet (Beta vulgaris L.), J.Exp. Bot. 44:1687-1694). Because the bulk of beet sucrose is localizedin the vacuoles of the cell, the (injury-)induced vacuolar invertaseplays a central role in storage sucrose loss.

At present there is no satisfactory solution to the problem of storagesucrose losses (Burba, 1976). The most important practices in the priorart consist in maintaining low temperatures (below 12° C.) and awell-defined atmospheric humidity (between 90 and 96%). All practicesused up to now to reduce the storage losses are, however,unsatisfactory.

Conversion of sucrose to the hexoses glucose and fructose in storage,and thus loss of sucrose, also occurs during the “cold sweetening” ofpotatoes. As a result of cold processing, a vacuolar invertase isinduced in the potato tubers and determines the ratio of sucrose tohexoses (Zrenner, R., Schüler, K., and Sonnewald, U. (1996), “SolubleAcid Invertase Determines the Hexose-to-Sucrose Ratio in Cold-StoredPotato Tubers,” Planta 198:246-252). The formation of hexoses as aresult of cold sweetening leads to diminutions of quality in the makingof, for example, French-fried potatoes.

Tomato fruits (Lycopersicon esculentum Mill.) exhibit a high watercontent. This is due in part to the osmotically active endogenous sugars(sucrose and hexoses). Lowering the total sugar content by means ofinhibiting the invertase-mediated hydrolysis of sucrose leads to smallerfruits with lower water content (Klann, E. M., Hall, B., and Bennett, A.B. (1996), “Antisense Acid Invertase (TIV1) Gene Alters Soluble SugarComposition and Size in Transgenic Tomato Fruit,” Plant Physiology112:1321-1330). Reducing the water content of the tomato fruits leads toa saving in energy costs for the production of fruit concentrates (e.g.,ketchup). Because the reduction of vacuolar invertase activity viainvertase antisense expression is incomplete because of the occurrenceof a variety of isoforms, the transgenic introduction of an invertaseinhibitor might result in great advantages, in particular if saidinvertase inhibitor has an equal inhibiting action on these variousisoforms.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to create a new system thatcauses essentially no sucrose storage-related losses in plants.

This object is achieved by virtue of the subject matters of theinvention characterized in the Claims.

DETAILED DESCRIPTION

A first subject matter of the invention relates to a nucleic acid thatcontains at least one nucleic acid sequence coding for a polypeptide,which polypeptide is capable of reducing or lowering the enzymaticactivity of an invertase.

The terms “nucleic acid” and “nucleic acid sequence” denote natural orsemisynthetic or synthetic or modified nucleic acid molecules fromdeoxyribonucleotides and/or ribonucleotides and/or modified nucleotides.

The term “polypeptide” denotes naturally occurring polypeptides andrecombinant polypeptides. Recombinant polypeptides denote a constructprepared by molecular-biological techniques, based on the natural DNA ofthe original genome or the natural DNA modified with a foreign DNAsequence, which construct can be recombined, for example with plasmids,and replicated and expressed in a suitable host system.

The expression “a polypeptide capable of reducing the enzymatic activityof an invertase” denotes a polypeptide that, in the process of bindingto an invertase, reduces the enzymatic activity of said invertase,complete inhibition being possible if there is a sufficient quantity ofthe inhibitor protein. A roughly 90% inhibition of the vacuolarinvertase is preferably to be achieved by means of the inhibitorexpression in the transgenic plant.

In a embodiment of the invention, the invertase in a plant cell isvacuolarly localized. In another embodiment, the invertase is localizedin the cell wall. In a further embodiment, the invertase is localized inthe cytosol. The invertase is preferably derived from sugar beet, potatoor tomato.

In a preferred embodiment of the invention, the nucleic acid comprisesthe nucleic acid sequences shown in FIGS. 1(a)-1(d) (SEQ ID No. 1), 3(SEQ ID No. 2), 12 (SEQ ID No. 3) and 14(a)-(b) (SEQ ID No. 4) orsegments or fragments thereof as well as nucleic acid sequences that canhybridize with the complementary sequences of the nucleic acid sequencesshown in FIGS. 1(a-(d), 3, 12 or 14(a)-(b) or segments or fragmentsthereof.

In another embodiment, the nucleic acid according to the inventioncontains a further nucleic acid sequence coding for a targetingsequence. The term “targeting sequence” denotes an amino acid sequencethat mediates cellular targeting into a well-defined cellularcompartment, for example targeting into the vacuoles.

In a preferred embodiment of the invention, the targeting sequencecomprises the vacuolar targeting sequence of barley lectin having thefollowing amino acid sequence:

SEQ ID NO: 9 LEGVFAEIAASNSTLVAE

In another embodiment, the nucleic acid according to the inventioncontains a further nucleic acid sequence coding for a signal peptide.The term “signal peptide” denotes a hydrophobic amino acid sequence thatis recognized by the signal recognition particle (SRP). The SRP mediatesthe synthesis of the entire polypeptide on the rough endoplasmicreticulum (ER), with the consequence that the resulting polypeptide isreleased into the ER lumen.

In a further embodiment, the nucleic acid according to the inventioncontains a nucleic acid sequence coding for an ER retention sequence.

In a preferred embodiment, the signal peptide is derived from aninvertase, preferably from cell-wall invertase from tobacco.

In another embodiment of the invention, the nucleic acid contains afurther nucleic acid sequence that comprises a promoter suitable forexpression in plants. This promoter or promoter sequence is preferablyderived from the same plant as the invertase. In an especially preferredembodiment of the invention, the promoter is a promoter specific topotato or sugar beet.

In summary, the nucleic acid according to the invention can comprise theabove-defined nucleic acid sequence coding the polypeptide and, ifappropriate, the above-defined nucleic acid sequence coding a targetingsequence and/or the above-defined promoter, where all nucleic acidsequences coding an amino acid sequence are preferably arranged in thereading frame and can be degenerated in accordance with the geneticcode.

A further subject matter of the invention is a vector that contains theabove-defined nucleic acid according to the invention for the expressionof the recombinant polypeptide in prokaryotic or eukaryotic host cells.The vector according to the invention can preferably contain suitableregulatory elements such as promoters, enhancers, termination sequences.The vector according to the invention can be, for example, an expressionvector or a vector for the preferably stable integration of the nucleicacid according to the invention in to the genetic material of a hostcell. A suitable expression system comprises, for example, the Tiplasmid or a binary plasmid system in Agrobacterium tumefaciens asvector for the stable integration of the nucleic acid according to theinvention into the genetic material of a plant. Further, the nucleicacid according to the invention can, for example, also be inserted intothe genetic material of a plant by means of the Ri plasmid ofAgrobacterium rhizogenes, by means of direct gene transfer viapolyethylene glycol, by means of electroporation, or by means ofparticle bombardment.

A further subject matter of the invention is a host cell that containsthe nucleic acid according to the invention or the vector according tothe invention. Suitable host cells are, for example, prokaryotes such asE. coli or eukaryotic host cells such as Saccharomyces cerevisiae,Schizosaccharomyces pombe, Hansenula polymorpha, Pichia pastoris andbaculovirus-infected insect cells.

A further subject matter of the invention is the polypeptide itself thatis coded by the above-defined nucleic acid sequence, where the nucleicacid sequence can be degenerated in accordance with the genetic code.The polypeptide according to the invention contains at least one aminoacid sequence segment capable of reducing the enzymatic activity of aninvertase. In an especially preferred embodiment, the polypeptidecomprises the amino acid sequences shown in FIGS. 1(a)-1(d) (SEQ ID No.5), 3 (SEQ ID No. 6), 12 (SEQ ID No. 7) and 14(a)-14(b) (SEQ ID No. 8)or segments or fragments thereof. The term “polypeptide” furthercomprises, for example, iso forms from the same plant as well ashomologous inhibitor sequences of other plant species, the homology atthe protein level preferably being >70%.

In a embodiment of the invention, the polypeptide further contains anamino acid sequence arranged at the C-terminus of the polypeptide, whichamino acid sequence comprises an above-defined targeting sequence and/oran ER retention sequence, for example “KDEL,” and/or an amino acidsequence arranged at the N-terminus of the polypeptide, which amino acidsequence comprises an above-defined signal peptide.

The nucleic acid sequence according to the invention, the vectoraccording to the invention, and the polypeptide according to theinvention can be prepared by means of prior art methods.

A further subject matter of the invention is a transgenic plant thatcontains at least the above-defined nucleic acid according to theinvention.

The term “transgenic plant” or “plant” comprises the entire plant assuch as well as its parts, such as root, stem, leaf, organ-specifictissue or cells, its reproductive material, in particular seeds, and itsembryos. This term further comprises starchy tubers and starchy roots,for example potato, sweet potato and cassava, and sugar plants, forexample sugar cane and sugar beet, as well as tomato and maize.

In a preferred embodiment of the invention, the wild type of thetransgenic plant is a sugar beet, a tomato or a potato.

A further subject matter of the invention relates to a method ofpreparing the transgenic plant according to the invention, wherein aplant cell is transformed by means of stable integration of theabove-defined nucleic acid into the genetic material and the transformedplant cell is regenerated to the transgenic plant.

Methods of preparing transgenic plants are known in the prior art.

A further subject matter of the invention relates to the use of theabove-defined nucleic acid for the preparation of a transgenic planthaving reduced storage sucrose loss.

It can be stated according to the invention that the reduction instorage sucrose losses by means of the expression of the above-definedpolypeptide as “invertase inhibitor protein” in transgenic plantsrepresents, surprisingly, a highly specific, environmentally safe methodfor improving the quality of, for example, sugar beets or potato tubers.For sugar beet, a reduction in required production capacity is madepossible by means of the boost in the efficiency of sugar recovery for agiven level of productiveness. In the case of potato, the productquality of potatoes, in particular for the making of French-friedpotatoes, is enhanced by means of the reduction in cold-induced hexoseformation. In the case of tomato, the water content of the tomato fruitis lowered by means of the reduction of osmotically active hexoses.

By means of the combination of the nucleic acid sequence encoding theinvertase inhibitor with a nucleic acid sequence encoding a suitabletargeting sequence, correct vacuolar targeting of the expressedinvertase inhibitors into the vacuoles can, for example, be achieved andthus the expression of the invertase inhibitor can be restricted inspace. Further, the expression of the invertase inhibitor can berestricted in time by means of the use of promoters specific to, forexample, beet or tuber.

BRIEF DESCRIPTION OF THE FIGURES

The Figures show the following:

FIGS. 1(a)-1(d) shows the cDNA from Nicotiana tabacum encoding theinvertase inhibitor, having a length of 1701 bp, the open reading frame(ORF) comprising 477 bp with starting nucleotide 1. The invertaseinhibitor coded by this nucleic acid sequence exhibits 159 amino acidswith a calculated molecular weight M_(r) of 18915 and a calculatedisoelectric point of 10.13.

FIG. 2 shows the schematic preparation of the inhibitor construct of apreferred embodiment of the invention for the transformation of plants.

FIG. 3 shows a further cDNA coding for an invertase inhibitor localizedin the cell wall of tobacco cells, having a length of 631 bp (exclusiveof poly(A)). The signal sequence used for secretion into the cell wallis marked italicized. The site of cleavage, which is identical to thepartially sequenced N-terminus of the mature protein, is marked by meansof an arrow.

FIG. 4 shows the expression of the recombinant tobacco invertaseinhibitor in E. coli. The cDNA shown in FIG. 3 was cloned into the pQEvector (Qiagen, Hilden, Germany). The recombinant protein was expressedas a His-tagged fusion protein. 4A: M, molecular-weight marker; 1:bacteria, noninduced; 2: bacteria induced with IPTG; 3: recombinanttobacco invertase inhibitor purified by affinity chromatography(Ni-NTA). 4B: Western Blot analysis of fractions 1-3 from A with apolyclonal antiserum directed against the inhibitor.

FIG. 5 shows dose-dependent inhibition of the cell-wall invertase fromtobacco by means of the recombinant inhibitor protein. The circles showinhibition after preincubation of both proteins without sucrose; thesquares show inhibition without preincubation.

FIG. 6 shows the induction of acid invertase activity in sugar beetsafter injury.

FIG. 7 shows inhibition of total invertase activity from injured sugarbeets by means of invertase inhibitor derived from tobacco cellcultures.

FIG. 8 shows inhibition of cell-wall invertase from sugar beet by meansof the recombinant tobacco invertase inhibitor (see FIGS. 3-5). Thecircles show inhibition after preincubation of both proteins withoutsucrose; the squares show inhibition without preincubation.

FIG. 9 shows inhibition of total invertase activity from injured sugarbeets (vacuolar invertase+cell-wall invertase) by means of therecombinant tobacco invertase inhibitor (see FIGS. 3-5). The circlesshow inhibition after preincubation of both proteins without sucrose;the squares show inhibition without preincubation.

FIG. 10 shows the immunological identification of vacuolar invertase(VI) from tomato fruits, as well as the detection of a tomato inhibitor(INH) homologous to the tobacco invertase inhibitor. Both proteins weredetected with polyclonal, monospecific antisera. After SDS-PAGE andWestern Blot, the VI shows two cleavage products of 52 and 20 kDa. TheVI binds completely to concanavalin A sepharose, whereas the tomatoinvertase inhibitor is present in roughly equal quantities of conA-binding and con A-nonbinding fractions.

FIG. 11 shows inhibition of the tomato VI by means of the recombinanttobacco invertase inhibitor (see FIGS. 3-5). The circles show inhibitionafter preincubation of both proteins without sucrose; the squares showinhibition without preincubation.

FIG. 12 shows the sequence of a partial cDNA of the tomato invertaseinhibitor, which was amplified from tomato flower cDNA by RT-PCR.

FIG. 13 shows a comparison of two (identical) partial tomato invertaseinhibitor clones with the tobacco invertase inhibitor (see FIG. 3).

FIGS. 14(a)-(b) shows the cDNA sequence of a cytosolic homolog of theinvertase inhibitor clone of FIG. 3. The protein encoded by this cloneis capable of inhibiting cytosolic invertases.

The invention is explained in more detail by means of the example thatfollows.

EXAMPLE

All methods used in the following example for the preparation of therequired gene constructs correspond to standard methods for work inmolecular biology (Ausubel, F., Brent, R., Kingston, R. E., Moore, D.D., Seidmann, J. G., Smith, J. A., and Struhl, K. (1987-1996), CurrentProtocols in Molecular Biology, Greene Publishing). The procedure can besubdivided into essentially the following steps:

(1) The inhibitor protein is purified to homogeneity by selective saltelution of the cell-wall protein, twofold ion-exchange chromatography,and subsequent SDS polyacrylamide gel electrophoresis.

(2) The homogeneous inhibitor protein is subjected to tryptic digestion,and the resulting peptides of the inhibitor protein are sequenced byEdman degradation.

(3) On the basis of the peptide sequences obtained, degenerate primersare synthesized; with their help, DNA fragments of the inhibitor cDNAare amplified from the overall cDNA by PCR.

(4) A cDNA library is prepared from tobacco cell cultures (in anexpression vector: Stratagene ZAP Express®).

(5) The resulting partial sequences of inhibitor cDNA (see step 2) areused for screening the cDNA library.

(6) The obtained full-length clone, after expression in E. coli (cloninginto the Qiagen pQE vector), is confirmed as to its function (invertaseinhibition).

(7) The segment of the cDNA clone coding for the inhibitor protein(FIG. 1) is amplified by PCR. Primers having restriction cleavage-sitesthat permit subsequent ligation with the signal sequence and thetargeting sequence are used for this purpose. The signal sequence isligated to the 5′ end, while the targeting sequence for the vacuoles isligated to the 3′ end. Recovery of signal sequence: The signal sequenceis amplified from the cDNA of the tobacco cell-wall invertase (Greiner,S., Weil, M., Krausgrill, S., and Rausch, T. (1995), Plant Physiology108:825-826) by PCR (region Met¹-Val²³). Primers having restrictioncleavage-sites that permit subsequent ligation to the inhibitor cDNA areused for this purpose. Recovery of targeting sequence: The targetingsequence is amplified from the cDNA for barley lectin (Bednarek, S. Y.,and Taikhel, N. V. (1991), Plant Cell 3:1195-1206). Again, primershaving restriction cleavage-sites that permit subsequent ligation to theinhibitor cDNA are used for this purpose. For the sense cloning of thenucleic acid shown in FIG. 3 (SEQ ID No. 2), the entire nucleic acid isexcised from the pBK-CMV vector (which is generated by in vivo excisionfrom the Stratagene ZAP Express phages) with the help of the restrictionendonucleases BamHI and XbaI. The DNA fragment obtained is now ligatedinto a BamHI/XbaI cleaved binary transformation vector and thentransformed into bacteria. For the antisense cloning of the nucleic acidshown in FIG. 3 (SEQ ID No. 2), the restriction endonucleases BamHI andKpnI are employed. Otherwise, the procedure is the same as in the sensecloning. An analogous procedure is used for the sense and antisensecloning of the nucleic acid from FIGS. 14(a)-14(b) (SEQ ID No. 4). Theconstructs thus obtained are used for Agrobacterium tumefaciens-mediatedgene transfer into plants (sugar beet, potato and tomato in theexample). The insertion of the vacuolar targeting sequence for thenucleic acids from FIGS. 3 and 4 is carried out as described for thenucleic acid from FIGS. 1(a)-1(d).

(8) The 5′ end of the gene construct cited in (7) is ligated to abeet-specific promoter, and the resulting construct is cloned into abinary expression vector.

(9) The target plant is transformed by a suitable prior arttransformation method. The structure of the gene construct used for thetransformation is shown in FIG. 2.

An antiserum against the invertase inhibitor expressed in tobacco cellswas developed for screening a cDNA library. A homologous cDNA probe wasalso obtained by PCR reactions with oligonucleotides derived frompartial amino acid sequences. Furthermore, a screening witholigonucleotides was performed. The clone in FIGS. 1(a)-1(d) wasisolated with the oligonucleotide screening. A 300 bp fragment wasamplified by RT-PCR and then employed as a probe for screening the cDNAlibrary. The clone in FIG. 3 was isolated in this way. The latter wasexpressed in E. coli as a His-tagged fusion protein (FIG. 4). Therecombinant inhibitor protein inhibits the cell-wall invertase fromtobacco, a partial protection of the substrate being observed for thisinvertase iso form (FIG. 5), said protection not occurring, however,with other vacuolar invertases or invertases localized in the cell wall(see below). What is more, a homolog to the inhibitor clone shown inFIG. 3, localized in the cytosol, was isolated (FIGS. 14(a)-14(b) ). Theprotein coded by this clone can act as an inhibitor for cytosolicinvertases.

The invertase activity in injured sugar beets (FIG. 6) can be inhibitedby the inhibitor protein isolated from tobacco cells (FIG. 7). Enzymekinetics with recombinant tobacco inhibitor protein confirm that boththe total invertase activity (FIG. 9) in injured sugar beets and thepartially purified cell-wall invertase of sugar beet (FIG. 8) can beinhibited by the invertase inhibitor from tobacco.

In tomato fruits, vacuolar invertase is primarily expressed, which isdegraded into two cleavage products (52 and 20 kDa, see FIG. 10) uponSDS-PAGE separation. Along with the vacuolar invertase, an invertaseinhibitor of approximately 19 kDa, presumably localized in the cell-wallspace, is also expressed; it cross-reacts with the antiserum against thetobacco invertase inhibitor (FIG. 10). The vacuolar invertase isolatedfrom tomato fruits is likewise inhibited by the recombinant tobaccoinvertase inhibitor (FIG. 11). The striking sequence homology of atomato cDNA partial sequence obtained by RT-PCR with the sequence of thetobacco invertase inhibitor (FIGS. 12 and 13) suggests that the tomatoinvertase inhibitor expressed in fruits might be compartmentalized (inthe cell-wall space) with respect to the vacuolar invertase and thusdoes not inhibit the vacuolar invertase in vivo.

In summary, the apoplastic tobacco invertase inhibitor (FIG. 3) has beendemonstrated to be capable of completely inhibiting both cell-wallinvertases and vacuolar invertases, in particular of sugar beet andtomato. Given correct cellular targeting, the tobacco invertaseinhibitor can thus be used in transgenic plants (sugar beet, potato,tomato) to reduce vacuolar invertases and/or invertases localized in thecell wall. The invertase inhibitor localized in the cytosol (FIGS.14(a)-14(b) ) regulates cytosolic invertases. Their inhibition intransgenic plants can also have an advantageous effect on thesucrose/hexose ratio.

What is claimed is:
 1. An isolated nucleic acid comprising at least onenucleic acid sequence coding for a polypeptide capable of reducing theenzymatic activity of an invertase, said nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 2; SEQ ID NO: 3; and SEQ ID NO:4.
 2. The nucleic acid according to claim 1, wherein said invertase islocalized in a plant cell.
 3. The nucleic acid according to claim 2,wherein said invertase is further localized in a plant cell at a siteselected from the group consisting of a vacuole of said plant cell, awall of said plant cell, and the cytosol of said plant cell.
 4. Thenucleic acid according to claim 1, wherein said invertase is selectedfrom the group consisting of sugar beet, potato, and tomato invertases.5. The nucleic acid according to claim 1, further comprising at leastone nucleic acid sequence coding for a signal peptide.
 6. The nucleicacid according to claim 5, wherein said signal peptide is an invertasesignal peptide.
 7. The nucleic acid according to claim 5, wherein saidsignal peptide is isolated from cell-wall invertase from tobacco.
 8. Thenucleic acid according to claim 1, further comprising at least onenucleic acid sequence coding for an endoplasmic reticulum retentionsequence.
 9. The nucleic acid according to claim 1, further comprisingat least one nucleic acid sequence, said sequence comprising a promotersuitable for expression in plants.
 10. The nucleic acid according toclaim 9, wherein said promoter and said invertase are isolated from thesame plant species.
 11. The nucleic acid according to claim 9, whereinsaid promoter is specific to a plant selected from the group consistingof potato, tomato, and sugar beet.
 12. A nucleic acid comprising: (a) afirst nucleic acid sequence, called an inhibitor sequence, coding for apolypeptide inhibiting the enzymatic activity of an invertase, saidsequence comprising a sequence selected from the group consisting of SEQID NO: 2; SEQ ID NO: 3; and SEQ ID NO: 4; (b) a second nucleic acidsequence, called a targeting sequence, coding for a targeting sequence;(c) a third nucleic acid sequence, called a signal sequence, coding fora signal peptide; and (d) a fourth nucleic acid sequence, called apromoter sequence, comprising a promoter sequence controlling expressionof each of said nucleic acid sequences; wherein said first, second,third, and fourth nucleic acid sequences are operatively linked in theorder promoter sequence, signal sequence, inhibitor sequence, andtargeting sequence such that expression of said sequences provides apolypeptide capable of inhibiting the enzymatic activity of aninvertase.
 13. A nucleic acid vector comprising a nucleic acid sequenceaccording to claim 1, said nucleic acid sequence being operativelylinked to an expression control sequence.
 14. The nucleic acid vectoraccording to claim 13, wherein said vector is a plasmid selected fromthe group consisting of the Ti plasmid of Agrobacterium tumefaciens, abinary plasmid system of Agrobacterium tumefaciens, and the Ri plasmidof Agrobacterium rhizogenes.
 15. A host cell which is a recombinant cellthat has been transformed with a recombinant nucleic acid, saidrecombinant nucleic acid having at least one nucleic acid sequenceaccording to claim
 1. 16. A transgenic plant comprising at least onerecombinant nucleic acid, said recombinant nucleic acid comprising atleast one nucleic acid according to claim
 1. 17. The transgenic plantaccording to claim 16, wherein said plant is isolated from a wild typeplant selected from the group consisting of plants having starchytubers, plants having starchy roots, potatoes, sweet potatoes, cassava,sugar plants, tomato, and maize.
 18. A method of producing a transgenicplant from a wild type plant, said method comprising the steps: (a)isolating a nucleic acid, said nucleic acid comprising a nucleic acidsequence coding for a peptide that inhibits the enzymatic activity of aninvertase, said nucleic acid selected from the group consisting of SEQID NO:2; SEQ ID NO:3; and SEQ ID NO:4; (b) transforming a plant cell ofsaid wild type plant by stably integrating said nucleic acid into thegenome of said plant cell; and (c) regenerating said plant cell toproduce a transgenic plant.
 19. The method according to claim 18,wherein said wild type plant is selected from the group consisting ofsugar beet, potato, tomato, and sugar cane.
 20. The transgenic plantaccording to claim 17, wherein said sugar plants are sugar cane or sugarbeets.
 21. An isolated nucleic acid sequence comprising a nucleic acidsequence coding for a polypeptide that reduces the enzymatic activity ofan invertase, said nucleic acid sequence selected from the groupconsisting of (a) SEQ ID NO. 2; (b) SEQ ID NO. 3; (c) SEQ ID NO. 4; and(d) nucleic acid sequences encoding polypeptides, said polypeptideshaving at least 70% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO. 6; SEQ ID NO. 7; and SEQ ID NO. 8;wherein said nucleic acid sequence, when introduced into an expressionvector and expressed in E. coli, produces a polypeptide capable ofinhibiting the enzymatic activity of an invertase.
 22. An isolatednucleic acid comprising a nucleic acid sequence coding for a polypeptidethat reduces the enzymatic activity of an invertase, said polypeptidehaving at least 70% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO. 6; SEQ ID NO. 7; and SEQ ID NO. 8;wherein said nucleic acid sequence, when introduced into an expressionvector and expressed in E. coli, produces said polypeptide capable ofinhibiting the enzymatic activity of an invertase.